Cancers recurring after chemotherapeutic treatment often present resistances to most of the currently available anti-cancer drugs. Similar resistances also occur in the chronic treatment of infectious diseases like HIV-AIDS. Such insensitivities to therapies pose immense problems to the treatment of the affected patients. One cause of multidrug resistances is the overproduction of a membrane protein, P-glycoprotein (P-gp). This member of the ABC-transporter family pumps chemotherapeutics out of cells and thereby lowers the effective concentration to sub-therapeutic levels. More than 30 years of research geared at finding effective inhibitors of P-glycoprotein that could be used as co-therapeutics to in the treatment of patients with resistant cancers has not yielded success. In previous work, we used ultrahigh throughput computational screening of very large drug-like compound databases and a structural model of P-glycoprotein to find novel molecules that inhibit the pump and may be developed into co-therapeutics to treat chemotherapy-resistant cancers. With these techniques and subsequent biochemical and biophysical assays, we identified four compounds out of several million that indeed blocked steps in the catalytic cycle of P- glycoprotein that are necessary for drug export. Three of these compounds reversed chemotherapy resistance in multidrug resistant prostate cancer cells in culture, while showing low to no toxicity to noncancerous cells. These compounds may serve as pharmacological lead compounds for the development of co-therapeutics for therapy resistant cancer or HIV-infected patients. We propose here enhancements to our computational screening methods to increase our prediction rate and the quality of identified P-gp inhibitors. This will be accomplished by taking into account the structural changes that the pump undergoes during transport. Identified potential inhibitors will be evaluated in biochemical assays for their efficacy of blocking P-glycoprotein action. Successful candidates will then be further evaluated for their potential to reverse multidrug resistance in cancer cells and their toxicity to normal cells in culture. We further propose to develop biophysical methods that will allow us to determine the molecular mechanism that leads to inhibition of P-glycoprotein by the discovered molecules. Identified novel P-glycoprotein inhibitors from our earlier studies, as well as those discovered in the proposed studies, will be further optimized in the future for pharmaceutical use.